Homing endonucleases are enzymes that catalyze DNA sequence specific double-strand breaks and can significantly stimulate homologous recombination at these breaks in cells. These enzymes have great potential for applications such as gene correction in gene therapy or gene alteration in systems biology, functional genomics, stem cell engineering, and metabolic engineering. However, homing endonucleases have a limited natural repertoire of target sequences, which severely hampers their applications. The broad and long-term goal of this research is to engineer homing endonucleases that recognize and cleave novel DNA sequences that are linked with human genetic disorders and to investigate the molecular determinants of the exquisite DNA sequence specificity of homing endonucleases. Using a well-characterized homing endonuclease I-SceI as a model system, we will use combinatorial protein engineering strategies to generate I-SceI variants that cleave new target DNA sequences found in a mutant hemoglobin beta gene associated with sickle cell anemia. Such engineered I-SceI mutants hold tremendous potential as novel gene therapy agents for sickle cell anemia. In addition, we will use a variety of biochemical and biophysical methods to characterize select engineered I-SceI variants both in vitro and in mammalian cells. Such studies will provide novel insights into the molecular basis of the homing endonuclease DNA sequence specificity. Equally important, we anticipate that this exploratory/developmental (R21) project will establish a technology platform for engineering a wide variety of DNA-modifying enzymes for biomedical research and human gene therapy.